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Ciliopathy patient variants reveal organelle-specific functions for TUBB4B in axonemal microtubules.Science (New York, N.Y.) Apr 2024Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes...
Tubulin, one of the most abundant cytoskeletal building blocks, has numerous isotypes in metazoans encoded by different conserved genes. Whether these distinct isotypes form cell type- and context-specific microtubule structures is poorly understood. Based on a cohort of 12 patients with primary ciliary dyskinesia as well as mouse mutants, we identified and characterized variants in the isotype that specifically perturbed centriole and cilium biogenesis. Distinct variants differentially affected microtubule dynamics and cilia formation in a dominant-negative manner. Structure-function studies revealed that different TUBB4B variants disrupted distinct tubulin interfaces, thereby enabling stratification of patients into three classes of ciliopathic diseases. These findings show that specific tubulin isotypes have distinct and nonredundant subcellular functions and establish a link between tubulinopathies and ciliopathies.
Topics: Animals; Humans; Mice; Axoneme; Centrioles; Cilia; Ciliary Motility Disorders; Mutation; Protein Isoforms; Tubulin; Male; Female; Mice, Knockout
PubMed: 38662826
DOI: 10.1126/science.adf5489 -
Cell Nov 2023To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization,...
To understand the molecular mechanisms of cellular pathways, contemporary workflows typically require multiple techniques to identify proteins, track their localization, and determine their structures in vitro. Here, we combined cellular cryoelectron tomography (cryo-ET) and AlphaFold2 modeling to address these questions and understand how mammalian sperm are built in situ. Our cellular cryo-ET and subtomogram averaging provided 6.0-Å reconstructions of axonemal microtubule structures. The well-resolved tertiary structures allowed us to unbiasedly match sperm-specific densities with 21,615 AlphaFold2-predicted protein models of the mouse proteome. We identified Tektin 5, CCDC105, and SPACA9 as novel microtubule-associated proteins. These proteins form an extensive interaction network crosslinking the lumen of axonemal doublet microtubules, suggesting their roles in modulating the mechanical properties of the filaments. Indeed, Tekt5 -/- sperm possess more deformed flagella with 180° bends. Together, our studies presented a cellular visual proteomics workflow and shed light on the in vivo functions of Tektin 5.
Topics: Animals; Male; Mice; Axoneme; Cryoelectron Microscopy; Flagella; Microtubules; Semen; Spermatozoa; Proteome
PubMed: 37865089
DOI: 10.1016/j.cell.2023.09.017 -
Seminars in Cell & Developmental Biology Jan 2023Ciliogenesis is a complex multistep process used to describe assembly of cilia and flagella. These organelles play essential roles in motility and signaling on the... (Review)
Review
Ciliogenesis is a complex multistep process used to describe assembly of cilia and flagella. These organelles play essential roles in motility and signaling on the surface of cells. Cilia are built at the distal ends of centrioles through the formation of an axoneme that is surrounded by the ciliary membrane. As is the case in the biogenesis of other cellular organelles, regulators of membrane trafficking play essential roles in ciliogenesis, albeit with a unique feature that membranes are organized around microtubule-based structures. Membrane association with the distal end of the centriole is a critical initiating step for ciliogenesis. Studies of this process in different cell types suggests that a singular mechanism may not be utilized to initiate cilium assembly. In this review, we focus on recent insights into cilium biogenesis and the roles membrane trafficking regulators play in described ciliogenesis mechanisms with relevance to human disease.
Topics: Humans; Centrioles; Axoneme; Cilia; Microtubules; Flagella
PubMed: 35351373
DOI: 10.1016/j.semcdb.2022.03.021 -
Seminars in Cell & Developmental Biology Feb 2021A finely regulated system of airway epithelial development governs the differentiation of motile ciliated cells of the human respiratory tract, conferring the body's... (Review)
Review
A finely regulated system of airway epithelial development governs the differentiation of motile ciliated cells of the human respiratory tract, conferring the body's mucociliary clearance defence system. Human cilia dysfunction can arise through genetic mutations and this is a cause of debilitating disease morbidities that confer a greatly reduced quality of life. The inherited human motile ciliopathy disorder, primary ciliary dyskinesia (PCD), can arise from mutations in genes affecting various aspects of motile cilia structure and function through deficient production, transport and assembly of cilia motility components or through defective multiciliogenesis. Our understanding about the development of the respiratory epithelium, motile cilia biology and the implications for human pathology has expanded greatly over the past 20 years since isolation of the first PCD gene, rising to now nearly 50 genes. Systems level insights about cilia motility in health and disease have been made possible through intensive molecular and omics (genomics, transcriptomics, proteomics) research, applied in ciliate organisms and in animal and human disease modelling. Here, we review ciliated airway development and the genetic stratification that underlies PCD, for which the underlying genotype can increasingly be connected to biological mechanism and disease prognostics. Progress in this field can facilitate clinical translation of research advances, with potential for great medical impact, e.g. through improvements in ciliopathy disease diagnosis, management, family counselling and by enhancing the potential for future genetically tailored approaches to disease therapeutics.
Topics: Animals; Axonemal Dyneins; Cilia; Ciliary Motility Disorders; Eye Proteins; Forkhead Transcription Factors; Gene Expression Regulation; Genotype; Humans; Inheritance Patterns; Intracellular Signaling Peptides and Proteins; Mutation; Proteins; Quality of Life; Respiratory Mucosa; Signal Transduction
PubMed: 33279404
DOI: 10.1016/j.semcdb.2020.11.007 -
The World Journal of Men's Health Jan 2020Microtubules are the prime component of the cytoskeleton along with microfilaments. Being vital for organelle transport and cellular divisions during spermatogenesis and... (Review)
Review
Microtubules are the prime component of the cytoskeleton along with microfilaments. Being vital for organelle transport and cellular divisions during spermatogenesis and sperm motility process, microtubules ascertain functional capacity of sperm. Also, microtubule based structures such as axoneme and manchette are crucial for sperm head and tail formation. This review (a) presents a concise, yet detailed structural overview of the microtubules, (b) analyses the role of microtubule structures in various male reproductive functions, and (c) presents the association of microtubular dysfunctions with male infertility. Considering the immense importance of microtubule structures in the formation and maintenance of physiological functions of sperm cells, this review serves as a scientific trigger in stimulating further male infertility research in this direction.
PubMed: 30350487
DOI: 10.5534/wjmh.180066 -
Proceedings of the National Academy of... Jul 2023Cilia build distinct subdomains with variable axonemal structures to perform diverse functions in cell motility and signaling. In sensory cilia across species, an...
Cilia build distinct subdomains with variable axonemal structures to perform diverse functions in cell motility and signaling. In sensory cilia across species, an axoneme differentiates longitudinally into a middle segment with nine microtubule (MT) doublets and a distal segment with nine MT singlets that extends from the A tubules of the doublets. Here, we study axoneme differentiation in by analyzing the flagellar inner junction protein FAP20 and PCRG1 that connect A and B tubules in . The nematode CFAP-20 is restricted to the middle segment with doublets, and its loss disconnects A and B tubules. However, PCRG-1 is absent from most sensory cilia, and its deletion does not disrupt cilia. Ectopic introduction of PCRG-1 into cilia generated abnormal MT doublets in the distal segment and reduced intraflagellar transport and animal sensation. Thus, the absence of an inner junction protein prevents B-tubule extension, which contributes to axoneme differentiation and ciliary function.
Topics: Animals; Axoneme; Cilia; Chlamydomonas; Caenorhabditis elegans; Biological Transport; Microtubules; Flagella
PubMed: 37463209
DOI: 10.1073/pnas.2303955120 -
Annual Review of Cell and Developmental... Oct 2022Ciliates are a diverse group of unicellular eukaryotes that vary widely in size, shape, body plan, and ecological niche. Here, we review recent research advances... (Review)
Review
Ciliates are a diverse group of unicellular eukaryotes that vary widely in size, shape, body plan, and ecological niche. Here, we review recent research advances achieved with ciliate models. Studies on patterning and regeneration have been revived in the giant ciliate , facilitated by modern omics methods. Cryo-electron microscopy and tomography have revolutionized the structural study of complex macromolecules such as telomerase, ribozymes, and axonemes. DNA elimination, gene scrambling, and mating type determination have been deciphered, revealing interesting adaptations of processes that have parallels in other kingdoms of life. Studies of common eukaryotic processes, such as intracellular trafficking, meiosis, and histone modification, reveal conservation as well as unique adaptations in these organisms that are evolutionarily distant from other models. Continual improvement of genetic and molecular tools makes ciliates accessible models for all levels of education and research. Such advances open new avenues of research and highlight the importance of ciliate research.
Topics: Biology; Ciliophora; Cryoelectron Microscopy; RNA, Catalytic; Telomerase
PubMed: 35901313
DOI: 10.1146/annurev-cellbio-120420-020656 -
Sub-cellular Biochemistry 2022Cilia are tail-like organelles responsible for motility, transportation, and sensory functions in eukaryotic cells. Cilia research has been providing multifaceted...
Cilia are tail-like organelles responsible for motility, transportation, and sensory functions in eukaryotic cells. Cilia research has been providing multifaceted questions, attracting biologists of various areas and inducing interdisciplinary studies. In this chapter, we mainly focus on efforts to elucidate the molecular mechanism of ciliary beating motion, a field of research that has a long history and is still ongoing. We also overview topics closely related to the motility mechanism, such as ciliogenesis, cilia-related diseases, and sensory cilia. Subnanometer-scale to submillimeter-scale 3D imaging of the axoneme and the basal body resulted in a wide variety of insights into these questions.
Topics: Axoneme; Cilia; Flagella
PubMed: 36151386
DOI: 10.1007/978-3-031-00793-4_15 -
Cell Oct 2019The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells. In humans, impaired axoneme function causes a range of ciliopathies. Axoneme...
The axoneme of motile cilia is the largest macromolecular machine of eukaryotic cells. In humans, impaired axoneme function causes a range of ciliopathies. Axoneme assembly, structure, and motility require a radially arranged set of doublet microtubules, each decorated in repeating patterns with non-tubulin components. We use single-particle cryo-electron microscopy to visualize and build an atomic model of the repeating structure of a native axonemal doublet microtubule, which reveals the identities, positions, repeat lengths, and interactions of 38 associated proteins, including 33 microtubule inner proteins (MIPs). The structure demonstrates how these proteins establish the unique architecture of doublet microtubules, maintain coherent periodicities along the axoneme, and stabilize the microtubules against the repeated mechanical stress induced by ciliary motility. Our work elucidates the architectural principles that underpin the assembly of this large, repetitive eukaryotic structure and provides a molecular basis for understanding the etiology of human ciliopathies.
Topics: Axoneme; Cell Movement; Cilia; Ciliopathies; Cryoelectron Microscopy; Humans; Microtubule Proteins; Microtubules; Stress, Mechanical
PubMed: 31668805
DOI: 10.1016/j.cell.2019.09.030 -
Cell Nov 2021Dynein-decorated doublet microtubules (DMTs) are critical components of the oscillatory molecular machine of cilia, the axoneme, and have luminal surfaces patterned...
Dynein-decorated doublet microtubules (DMTs) are critical components of the oscillatory molecular machine of cilia, the axoneme, and have luminal surfaces patterned periodically by microtubule inner proteins (MIPs). Here we present an atomic model of the 48-nm repeat of a mammalian DMT, derived from a cryoelectron microscopy (cryo-EM) map of the complex isolated from bovine respiratory cilia. The structure uncovers principles of doublet microtubule organization and features specific to vertebrate cilia, including previously unknown MIPs, a luminal bundle of tektin filaments, and a pentameric dynein-docking complex. We identify a mechanism for bridging 48- to 24-nm periodicity across the microtubule wall and show that loss of the proteins involved causes defective ciliary motility and laterality abnormalities in zebrafish and mice. Our structure identifies candidate genes for diagnosis of ciliopathies and provides a framework to understand their functions in driving ciliary motility.
Topics: Amino Acid Sequence; Animals; Cattle; Cilia; Cryoelectron Microscopy; Dyneins; Embryo, Mammalian; Female; Male; Mammals; Mice, Inbred C57BL; Microtubule Proteins; Microtubules; Models, Molecular; Mutation; Proteins; Trachea; Zebrafish; Zebrafish Proteins; Mice
PubMed: 34715025
DOI: 10.1016/j.cell.2021.10.007